Transcription Regulation Of CYP321A1 In Helicoverpa ZEA

Cytochrome P450 monooxygenases (P450s) are a superfamily of heme-binding enzymes that play a paramount role in mediating plant-insect interactions. All plants use P450s to synthesize toxic defense compounds known as allelochemicals. Herbivorous insects, on the other hand, employ P450s to metabolize allelochemicals. Moreover, when feeding on plants, insect herbivores can continuously adjust the expression levels of their counterdefensive P450s to cope with escalating plant defenses by perceiving and transducing signals, particularly allelochemicals, from their host plants. Such an inducibility of P450s by allelochemicals is essential for the adaptation of insect herbivores to their host plant. However, how insects detect and transduce allelochemicals into elevated counterdefense phenotypes remains largely a mystery.To advance understanding of signal perception and transduction of plant allelochemicals in herbivorous insects, I choose to study the regulatory control of the P450 gene CYP321A1 in Helicoverpa zea, a polyphagous lepidopteran of economic importance. CYP321A1 is a typical counterdefensive P450 that metabolizes an array of allelochemicals and insecticides. Expression of CYP321A1 is constitutively low, but highly inducible upon exposure to a wide range of plant defensive allelochemicals including flavone and xanthotoxin.To define the cis-acting elements for flavone inducibility of the allelochemical- -metabolizing CYP321A1, functions of varying length of CYP321A1 promoter are examined in homologous H. zea fatbody cells by the Dual-Luciferase Reporter Assay System. Progressive 3’deletions reveal presence of positive elements in the 5’untranslated region (UTR). Progressive 5’deletions map out one essential element region, four enhancer regions, and two silencer regions. Further fine mapping by progressive 5’deletions localizes the essential basal and flavone inducible element to a 36 bp region from -109 to -74. This 36 bp essential element, designated as xenobiotic response element to flavone (XRE-Fla), contains a 5’AT-only TAAT inverted repeat (-109 to -100), a GCT mirror repeat (-99 to -93) and a 3’antioxidant response element-like (ARE-like) element (-92 to -74). Internal deletions and substitution mutations show that both the ARE-like element and the GCT repeat are necessary for the basal and flavone-induced expression, whereas the TAAT repeat is only necessary for the maximal flavone inducibility. Electrophoresis mobility shift assays demonstrate that XRE-Fla specifically binds to nuclear extracts from H. zea fatbody cells via its ARE-like element and flavone treatment increased the binding of nuclear factors to the XRE-Fla. These results demonstrate that the basal and flavone-inducible expression of CYP321A1 is regulated primarily by XRE-Fla and secondarily by other cis elements scattered in its promoter and 5’UTR.To determine if CYP321A1 gene responds to xanthotoxin, which is structurally different from flavone, via the same or different cis-elements, xanthotoxin-induced promoter activities of varying length of CYP321A1 promoter are examined in H. zea fatbody cells. Progressive 3’and 5’deletions map out four positive xanthotoxin response elements to the 5’untranslated region (5’UTR), -119 to -159, -278 to -310, and -310 to -558, respectively, two negative xanthotoxin response elements to -199 to -237 and -1218 to -1470, respectively, and one essential xanthotoxin response element to the same 36-bp region where XRE-Fla resides. Other than the XRE-Fla, 3 out of the above 6 regions are known to contain positive (5’UTR, -119 to -159) or negative (-1218 to -1470) flavone response elements. Further deletions and nucleotide substitution mutations show that the three components of the XRE-Fla regulate the xanthotoxin and flavone induction of CYP321A1 in the same manner. Mixtures of xanthotoxin plus flavone (1:1 ratio) exhibit an additive induction effect on the expression of CYP321A1. These data suggest that xanthtoxon- and flavone-induced expressions of CYP321A1 are mediated largely by the same set of cis elements and transcription factors. Dual luciferase assays demonstrate that diethyl maleate (DEM) and tert-butylhydroquinone (tBHQ), two ARE-Nrf2 (antioxidant response element /NF-E2-related factor 2)pathway markers, like flavone and xanthotoxin, induce the promoter activities of the wild type CYP321A1 promoter constructs. When the ARE-like element of the XRE-Fla is internally deleted, neither DEM nor tBHQ can increase the promoter activities of the corresponding ARE-deleted constructs. Fluorescent measurements of reactive oxygen species (ROS, a indicator of oxidative stress) with the molecular probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) confirm that exposure to DEM, xanthotoxin or flavone results in ROS burst in H. zea fatbody cells. The ROS burst elicited by flavone, xanthotoxin, or DEM is significantly reduced when H. zea fatbody cells are pretreated with the ROS production blocker diphenylene iodonium chloride [DPI, an inhibitor of NADPH oxidase (Nox), a key enzyme for ROS generation]. Further dual luciferase assays show that DPI pretreatment of H. zea fatbody cells transfected with the wild type CYP321A1 promoter constructs inhibits the induction of CYP321A1 promoter activity by flavone, xanthotoxin, and DEM. Ectopic expression of Drosophila melanogaster CncC (i.e. gain-of-function analysis) in H. zea fatbody cells increases the basal and induced promoter activities of the wild type CYP321A1 promoter constructs by flavone, xanthotoxin, and DEM. By contrast, RNAi (RNA interference) silencing of the endogenous H. zea CncC (i.e. loss-of-function analysis) reduces the basal and induced promoter activities of the wild type CYP321A1 promoter constructs by flavone, xanthotoxin, and DEM. The results suggest that CncC is crucial for transcriptional regulation of CYP321A1 in response to flavone, xanthotoxin or DEM. Taken together, these results demonstrate that the ARE-CncC signaling pathway, parallel to the vertebrate ARE-Nrf2 pathway, regulates allelochemical induction of CYP321A1 in H. zea.